Pharmaceutical Research, Vol. 23, No. 5, May 2006 (# 2006)
DOI: 10.1007/s11095-006-9933-1

Research Paper
Recombinant Human Serum Albumin Dimer has High Blood Circulation
Activity and Low Vascular Permeability in Comparison
with Native Human Serum Albumin
Sadaharu Matsushita,1 Victor Tuan Giam Chuang,1,2 Masanori Kanazawa,1 Sumio Tanase,3Keiichi Kawai,4 Toru Maruyama,1 Ayaka Suenaga,1 and Masaki Otagiri1,5Received September 8, 2005; accepted January 12, 2006
Purpose. Human serum albumin (HSA) is used clinically as an important plasma expander. Albumin
infusion is not recommended for critically ill patients with hypovolemia, burns, or hypoalbuminemia
because of the increased leakage of albumin into the extravascular spaces, thereby worsening edema. In
the present study, we attempted to overcome this problem by producing a recombinant HSA (rHSA)
dimer with decreased vascular permeability and an increased half-life.
Methods. Two molecules of rHSA were genetically fused to produce a recombinant albumin dimer
molecule. The pharmacokinetics and biodistribution of the recombinant proteins were evaluated innormal rats and carrageenin-induced paw edema mouse model.
Results. The conformational properties of this rHSA dimer were similar to those for the native HSA(the HSA monomer), as evidenced by the Western blot and spectroscopic studies. The biological halflife and area under the plasma concentrationYtime curve of the rHSA dimer were approximately 1.5
times greater than those of the monomer. Dimerization has also caused a significant decrease in the total
body clearance and distribution volume at the steady state of the native HSA. rHSA dimer accumulated
to a lesser extent in the liver, skin, muscle, and fat, as compared with the native HSA. Up to 96 h, thevascular permeability of the rHSA dimer was less than that of the native HSA in paw edema mousemodels. A prolonged plasma half-life of the rHSA dimer was also observed in the edema model rats.
Conclusions. rHSA dimer has a high retention rate in circulating blood and a lower vascularpermeability than that of the native HSA.
KEY WORDS: biodistribution; human serum albumin; pharmacokinetic analysis; recombinant humanserum albumin dimer; vascular permeability.

INTRODUCTION
Human serum albumin (HSA), the most abundant
protein in plasma, is responsible for 80% of the colloid
osmotic pressure of plasma (25Y33 mmHg). Because of this, it

1

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto
862-0973, Japan.
2
Department of Pharmacy, Faculty of Allied Health Sciences,
Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz,
50300 Kuala Lumpur Malaysia.
3
Department of Analytical Biochemistry, School of Health Sciences,
Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0976, Japan.
4
School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-1192, Japan.
5
To whom correspondence should be addressed. (e-mail: otagirim@
gpo.kumamoto-u.ac.jp
ABBREVIATIONS: AUC, area under the plasma concentrationYtime curve; CLtot, total body clearance; HSA, human serum
albumin; rHSA, recombinant HAS; Vdss, distribution volume of thesteady state; %ID, percentage of injected dose.
0724-8741/06/0500-0882/0 # 2006 Springer Science + Business Media, Inc.

is mainly used clinically to maintain colloid oncotic pressure
and to increase the circulating plasma volume (1). HSA
product was developed for the treatment of shock where
HSA Fraction V, USP, served as an attractive agent for thecorrection of hypovolemia, burns, and various diseases that
result in albumin loss. In spite of the many theoreticalbenefits of albumin infusion in critically ill patients, there
has been little evidence to support its widespread clinical use.
For many years, albumin infusion was believed to improvevital prognosis. Recently, a meta-analysis that included 24
studies involving 1419 patients published by Cochran Injuries
Group Albumin Reviewers suggested that the administrationof albumin-containing fluids resulted to a 1.68 times increasein the relative risk of death when compared to a crystalloid
solution (2). In contrast, no difference in mortality wasreported in a large-scale clinical trial in Australia and New
Zealand, in which 4% albumin was compared to 0.9%
sodium chloride for intravascular fluid resuscitation inpatients in intensive care units (ICUs) (3).
Reviews published on the parenteral use of albumin andthe consensus of several conferences recommended that theadministration of HSA can be justified for acute circulatory882

Biological Property of Recombinant HSA Dimer

883
sequence was a gift from Tonen Co. (Tokyo, Japan). The
restriction enzymes EcoRI, XhoI, AvaI, BamHI, and SalI
and E. coli JM109 were obtained from Takara Shuzo Co.,
Ltd. (Kyoto, Japan). KOD-Plus-DNA polymerase wasobtained from Toyobo Co., Ltd. (Osaka, Japan). The
restriction enzyme PciI was obtained from New England
Biolabs (Beverly, MA, USA). A DNA sequence kit was
obtained from Perkin-Elmer Applied Biosystems (Foster
City, CA, USA). The Pichia Expression Kit and therestriction enzyme NcoI were purchased from Invitrogen
Corp. (Carlsbad, CA, USA). [125I] Iodine-125 was purchasedfrom Amersham Biosciences Corp. (Piscataway, NJ, USA).
[111In] Indium chloride was supplied by Nihon Medi-Physics(Takarazuka, Japan). Trichloroacetic acid (TCA) was
purchased from Nakalai Tesque (Kyoto, Japan). 1Carrageenin was purchased from Sigma (St. Louis, MO,
USA). Other chemicals used were obtained from commercial
suppliers.

problems caused by hypovolemia, but it is seldom warranted
and is used merely to increase albumin concentrations incases of hypoalbuminemia (4). Only shock due to recent
blood loss is an indication of the use of parenteral albumin,
because the incidence of shock to acute-phase or hyperallergic reactions is accompanied by increased albumin flux
to the extravascular compartment and adding to the albuminpool in this case would only expand this compartment andbring on edema (5Y7). The lung is particularly susceptible toalbumin overloading, and albumin administration for respiratory distress has been a matter of debate because of thepossibility that pulmonary edema may result (8,9). Cardiac
failure is another hazard of an overzealous increase incirculatory volume (10).
It is well known that capillary protein permeability is
increased in many pathological and physiological conditions.
For instance, the rate of protein extravasation may increase
up to 100 times the normal rate in burn injury. Under suchconditions, the albumin molecules administered as a plasma
expander are transported to organs and cause a further
accumulation of fluids, hence worsening the disease. The
frequent administration of albumin infusion to maintain thedesired blood concentration, as well as the oncotic pressure,inevitably leads to the poor retention of albumin infused
during the disease state. Although albumin receptors havebeen reported to mediate the transport of albumin across thecellular membrane, the mechanism of leakage during inflammation may involve a different route of extravasation sincecytokines are known to loosen tight junctions (11).
In other words, if a form of albumin that possesses good
blood retention and low extravasation properties could be
developed, the frequency of administration could be decreased and further accumulation of fluids in tissues could be
prevented. Hence, increasing the molecular size of albumin
to stop leakage to extravascular spaces may be a potential
solution in making albumin a superior candidate as a volume
replacement therapeutic agent. This type of albumin would
not only be superior for clinical use but would also be usefulpharmacoeconomically.
In addition, since albumin is typically harvested using
conventional techniques involving the fractionation of plasma obtained from blood donors, possible viral/prion contaminants contained in the preparation impose risk to users.
As a solution to this problem, the development of recombinant technology in producing therapeutic proteins such as
HSA has been promoted. In this study, we genetically fused
two molecules of HSA and used Pichia pastoris to express therecombinant HSA (rHSA) dimer protein. The pharmacokinetic and pharmacological properties of the rHSA dimer
were then evaluated.

An rHSA dimer in which two HSA molecules are linked
with an amino acid linker (GGGGS)2 was designed. The
rHSA dimer expression vector was constructed through thefour-piece ligation of the following: (1) pPIC9K vector wasdigested with BamHI and EcoRI; (2) the fragment derived
from the digestion of pPIC9K with BamHI and XhoI
restriction sites was amplified by PCR; (3) the PCR producton the 50 side of the rHSA dimer, amplified using HSA
cDNA as the template, was digested with XhoI and AvaI(HSAcDNA-1); (4) the PCR product on the 30 side of rHSA
dimer, which was amplified using HSA cDNA as the
template, was digested with AvaI and EcoRI (HSAcDNA2). These four DNA fragments were separated from thecleavage products by agarose gel electrophoresis and purified.
Construction of the expression vector of the rHSA dimer
was achieved by a four-piece ligation (Fig. 1). The resulting
vector was transformed into E. coli JM109 and amplified.
The sequence of the rHSA dimer in the resulting vector was
confirmed by verifying the sequence of gel extract cDNA
fragments of the resulting vector digested by PciI or NcoI.
The resulting vector was introduced into the yeast P. pastoris(strain GS115), and the rHSA dimer was expressed andpurified. The purified rHSA dimer was confirmed by Nterminal amino acid sequences analysis, sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
and Western blot analysis. The structure of the rHSA dimer
was estimated from fluorescence and circular dichroism (CD)
spectral data (13).

MATERIALS AND METHODSTransformation, Expression, and Purificationof the rHSA Dimer

Materials
Native HSA was donated by the Chemo-Sera-Therapeutic
Research Institute (Kumamoto, Japan) and defatted bymeans of a charcoal treatment as described by Chen (12). A
chimeric plasmid (pJDB-ADH-L10-HAS-A) having cDNAfor the mature form of HSA along with an L10 leader

Synthesis, Purification, and Structure Analysis
of rHSA Dimer

Construction of the expression vector of the rHSA dimer was
made by a four-piece ligation (Fig. 1). The resulting vector
was transformed into E. coli JM109 and amplified. The
sequence of the rHSA dimer in the resulting vector was
confirmed on gel extracts of the cDNA fragments of theresulting vector digested by PciI or NcoI. The resulting
vector was linearized with SalI and introduced into the yeast

Matsushita et al.

884

Fig. 1. Schematic of the pPIC9K expression vector containing the rHSA dimer gene (A) and the sequence of theprimers used in the construction of the rHSA dimer (B). The restriction site sequences are underlined. The linker
regions are in boldface.

P. pastoris (strain GS115) by electroporation using a GENEPULSER II (Bio-Rad, Hercules, CA, USA) according to the
manufacturer’s instruction from Invitrogen. His+ colonies
were subcultured in yeast protein dextrose (YPD) and plated
on YPD agars containing 0.25Y3 mg/mL to select for
multicopy recombinants. A 1-mL overnight preculture of thebest expression recombinant yeast was used to inoculate 1 L ofglycerol complex medium (1% yeast extract, 2% peptone,
100 mM potassium phosphate buffer, pH 6.0, 1.34% yeast
nitrogen base, 4  10–5% biotin, 3% glycerol) and incubatedfor 2 days at 30-C in three 3-L baffle flasks. The cells were
harvested by centrifugation (1500  g, 10 min) and resuspended in a buffered 250-mL methanol complex medium (1%yeast extract, 1% peptone, 100 mM potassium phosphatebuffer, pH 6.0, 1.34% yeast nitrogen base, 4  10–5% biotin,
1% methanol) for 4 days at 30-C in twelve 1-L baffle flasks.
An additional 1% methanol was added at 12-h intervals. The
secreted rHSA dimer was purified on a Blue Sepharose CL-6B
column (Amersham Biosciences). Isolated protein was
defatted using the charcoal procedure described by Chen
(12), deionized, freeze dried, and then stored at –20-C until
use. N-terminal amino acid sequences of rHSA dimer were
determined using a Perkin-Elmer ABI 477A protein
sequencer.
Fluorescence and CD Spectra Measurements
Intrinsic fluorescence spectra of the proteins were measured using a Jasco FP-777 spectrofluorometer (Tokyo,

Japan) equipped with thermostatically controlled 1-cm
quartz cells and 5-nm excitation and emission bandwidths.
Native HSA and the rHSA dimer were excited at 295 nm,
and the spectra were corrected for buffer baseline fluorescence. CD spectra were obtained using a JASCO J-720
spectropolarimeter (JASCO, Tokyo, Japan) at 25-C. Farand near-UV CD spectra were recorded at protein concentrations of 5 2M (native HSA) and 2.5 2M (rHSA dimer) andat 15 2M (native HSA) and 7.5 2M (rHSA dimer),
respectively, in 67 mM phosphate buffer (pH 7.4).
SDS-PAGE and Western Blot Analysis
The rHSA dimer was analyzed via SDS-PAGE, using 8%
polyacrylamide gel, and detected by staining with Coomassie
blue. Western blotting was performed using an anti-HSA
antibody raised in a rabbit as the primary antibody followedby an antirabbit secondary antibody conjugated to horseradish peroxidase. Proteins were detected using an enhanced
chemiluminescence detection kit.
In Vivo Experiments: Animals
Male Wistar rats (5 weeks old, 150Y170 g) and male ddY
mice (6 weeks old, 28Y30 g) were purchased from the Kyudou
Co. (Kumamoto, Japan) and Japan SLC, Inc. (Shizuoka,
Japan), respectively. The animals were maintained under conventional housing conditions, with free access to water andBiological Property of Recombinant HSA Dimer
food throughout the experimental period, and allowed to acclimatize to the laboratory environment for 1 week. After 1
week (1 week before injection of labeled protein), rats injectedwith 111In-labeled protein were given water, which contains
5 mM sodium iodide, until the end of the experiment. All
animal experiments were undertaken according to theguideline principles and procedures of Kumamoto University
for the care and use of laboratory animals.
Radiolabeling with 125I
Native HSA and rHSA dimer were labeled with 125I using
the Iodo-gen (1,3,4,6-tetrachoro-3a,6a-diphenylglycoluri)
method (14). Nonincorporated 125I was removed using a
PD-10 column (Amersham Biosciences). 125I-labeled protein
was eluted from PD-10 column with phosphate-bufferedsaline (PBS). The derivative solution was concentrated andwashed with saline by ultrafiltration.
Radiolabeling with 111InFor the tissue distribution experiments, native HSA andrHSA dimer were radiolabeled with 111In using the bifunctional chelating agent diethylenetriaminepentaacetic
acid (DTPA) anhydride according to the method of
Hnatowich et al. (15). In a typical experiment, each sample(5 mg) was dissolved in 1 mL 0.1 M 4-(2-Hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) buffer, pH 7, andmixed with 15 mM DTPA anhydride in 10 2L dimethyl
sulfoxide. The mixture was stirred for 15 min at room
temperature, and the radiolabeled product was purified bygel filtration using a Sephadex G-25 column (Amersham
Biosciences) to remove unreacted DTPA. Fractions
containing the sample were collected and concentrated byultrafiltration at 4-C. A 20-2L aliquot of an 111InCl3 solution(37 MBq/mL) was then added to 20 2L of 0.1 M citratebuffer, pH 5.5, and 40 2L of a DTPA-coupled derivative
solution was added to the mixture. After 15 min, the mixture
was applied to a PD-10 column and eluted with 0.1 M citratebuffer, pH 5.5. The derivative fractions were collected andconcentrated by ultrafiltration at 4-C (16).

885(AUC) was calculated by fitting an equation to the plasma
precipitated by TCA concentrations of the derivatives using
MULTI, a nonlinear least-squares program (17).
Biodistribution Experiment
We injected 111In-protein into the tail vein of male ddY
mice. At 1, 4, 12, and 24 h after injection of these radiolabeledproteins, the percentage of the injected dose (%ID) per gramor milliliter of several tissues was estimated (13). Male ddY
mice received a 0.1-mg/kg dose (16.7  106 cpm/kg) of 111Innative HSA or 111In-rHSA dimer conjugate in saline byinjection into the tail vein. At 1, 4, 12, and 24 h after injectionof these radiolabeled proteins, blood was collected from thevena cava with the animal under ether anesthesia, andplasma was obtained by centrifugation. At each of thesetime points, an animal was killed for excision of kidney, liver,spleen skin, muscle, and fat. These organs were then rinsed
with saline and weighed, and the radioactivity contained by
the samples was determined. 111In radioactivity was counted
using a +-counter (ARC-5000; Aloka).
Carrageenin-Induced Paw Edema
Paw edema was induced by the subcutaneous injection of
50 2L of a saline solution containing 0.4% carrageenin into theright paw of male ddY mice, and 50 2L of saline solution alone
was injected to the left paw as a control. The time ofcarrageenin injection was designated as time 0. After the
carrageenin injection, the mice were injected with a 0.1-mg/kg
dose (16.7  106 cpm/kg) of 125I-native HSA or 125I-rHSA
dimer into a tail vein at different time points. The mice were
exsanguinated to death 15 min after the injection ofradiolabeled protein. Both paws were removed, and theradioactivity in each paw was measured. Footpad thickness
was measured twice immediately before the injection ofcarrageenin and the radiolabeled protein with a thickness

Pharmacokinetic Analysis
Iodination proteins (1.0  108 cpm/kg, 0.1 mg/kg) in saline
were injected into the tail vein of male Wistar rats. Blood(100 2L) samples were periodically collected from the tailvein using a heparinized needle at 1, 5, 10, 20, 30, and 45 min
and at 1, 2, 4, 8, 12, 18, 24, 36, 48, 60, 72, 84, and 96 h, andplasma was obtained by centrifugation. The radioactivity ofplasma (20 2L) samples was determined using a +-counter(ARC-380; Aloka, Tokyo, Japan); 1 mL of 1% bovine serum
albumin was then added to the plasma followed by 1 mL of
40% TCA. The sample was incubated with stirring for 10
min. After the incubation, the sample was centrifuged andthe radioactivity in the pellet was recounted on the +-counter.
Urine and feces were collected at 24-h intervals, and blood
was collected from other rats that were kept in a metabolic
cage. The area under the plasma concentrationYtime curve

Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(A) and Western blot analysis (B) of native HSA and rHSA dimer.
Lane 1, native HSA; lane 2, rHSA dimer (3 2g per lane).

Matsushita et al.

886
RESULTS
Cloning and Expression of rHSA Dimer

Fig. 3. Plasma level of radiolabeled proteins after intravenous
administration of 125I-native HSA (filled symbol) or 125I-rHSA
dimer (open symbol) to normal rats. Each point represents themean T SD (n = 4). %ID, percentage of injected dose.

gage 7301 (Mitutoyo, Kanagawa, Japan). Paw edema wasexpressed in terms of the difference in footpad thickness
between the right and the left paw.
Carrageenin Air Pouch
Inflammation of the carrageenin air pouch was induced
according to the method of Tsurufuji (18). Male Wistar rats,
6 weeks of age and weighing 180Y200 g, were subcutaneously
injected, under light ether anesthesia, with 8.0 mL of air on
the back to create an air pouch. After 24 h, 4.0 mL of a 2%(w/v) carrageenin solution in 0.9% NaCl was injected into the
air pouch that had been produced. The carrageenin solution
was autoclaved at 120-C for 20 min and injected, after
cooling, at a temperature of 40Y45-C. Immediately before theinjection, penicillin and streptomycin were added in aconcentration of 0.1 mg/mL each to the carrageenin solution.
After 6 days of carrageenin injection, 1.0  108 cpm/kg, 0.1 mg/
kg 125I-native HSA or 125I-rHSA dimer was injected into a
tail vein. The day of radiolabel protein injection was
designated as day 0. Collection of blood, plasma, urine, andfeces and a pharmacokinetic analysis followed the above
method (Pharmacokinetic Analysis). The weight and radioactivity of the pouch fluid was measured after exsanguinationof the rats to death.
Statistical Analysis
All data are presented as mean T SD. Significant differences
between native HSA and rHSA dimer were tested byStudent’s t test.

The rHSA dimer was expressed using P. pastoris. The
HSA sequence contains 585 amino acids. The rHSA dimer
was designed to contain two sets of the 585 amino acids fusedwith a linker of 10 amino acid (GGGGS)2. The polypeptide
linker is flexible and highly hydrophilic and, hence, isresistant to proteases (19,20). Construction of the expression
vector of the rHSA dimer was made by a four-piece ligation(Fig. 1). The reason for the four-piece ligation was that
pPIC9K cannot be used directly due to the presence of the
XhoI restriction site in the kanamycin resistance gene.
Furthermore, the use of other restriction enzymes would
result in the addition of some other amino acids in theexpressed protein. Although there is another alternative forusing pPIC9, which does not contain a kanamycin resistancegene, pPIC9K is easier than pPIC9 in the selection of a highexpression strain. The N-terminal amino acid sequence of therHSA dimer expressed in the present study was found to beconsistent with that of native HSA.
Structural Aspects of rHSA Dimer
Sodium dodecyl sulfate polyacrylamide gel electrophoresisof the rHSA dimer showed a single band, and the dimer
protein was recognized by an anti-HSA antibody. As shownin Fig. 2, the molecular weight of rHSA dimer (13 kDa) wasapproximately twice the molecular weight of native HSA in
SDS-PAGE. The intrinsic fluorescence spectra of Trp-214,
the only tryptophan residue of HSA, in the rHSA dimer was
slightly low compared with that of native HSA (data notshown). To obtain information on secondary and tertiary
protein structures, CD measurements were performed in the
far-UV and near-UV regions (data not shown). A little
decrease in the molar ellipticity of the rHSA dimer wasobserved in comparison with that of native HSA in the farUV CD spectrum. Although some spectral changes wereobserved, the near-UV CD spectra of the rHSA dimer show
the same minima and shape as that of native HSA. These
results suggest that the rHSA dimer structure is almost
identical to that of native HSA.
Pharmacokinetic Analysis of rHSA Dimer
Pharmacokinetics of the 125I-labeled native HSA andrHSA dimer was evaluated by residual TCA-precipitable
plasma radioactivity. Figure 3 shows the time courses ofplasma concentration of radiolabeled rHSA dimer and native
HSA, and the pharmacokinetic parameters for these two
proteins using a two-compartment model are listed in Table I.

Table I. Pharmacokinetic Parameters After an Intravenous Administration of 125I-native HSA or 125I-rHSA Dimer to Normal Rats

125

I-native HAS
I-rHSA dimer

125

AUC (min x %ID/mL)

CLtot (2L/h)

Vdss (mL)

T1/2, " (h)154.6 T 15.5
265.6 T 15.2.

654.6 T 65.0
380.1 T 25.6.

20.01 T 1.35
18.13 T 1.36

26.19 T 0.74
36.53 T 1.14*

All values are mean T SD (n = 4).
*p < 0.005, . p < 0.001 vs. 125 I-HSA native.

Biological Property of Recombinant HSA Dimer

887

Fig. 4. Tissue distribution of 111In-native HSA (filled bar) and 111In-rHSA dimer (open bar) at 1, 4, 12, and 24 h after an intravenous
administration to normal mice. Lanes 1 and 5, 1 h; lanes 2 and 6, 4 h; lanes 3 and 7, 12 h; lanes 4 and 8, 24 h. Each bar represents themean T SD (n = 3). *p < 0.05, .p < 0.01, -p < 0.005, `p < 0.001 vs. 125I-native HSA.

The AUC and half-life of the 125I-rHSA dimer were increased
by about 1.5 times, as compared with those of 125I-native
HSA. On the other hand, the 125I-rHSA dimer showed a twothirds decrease in total body clearance (CLtot), and a decline
in the distribution volume of the steady state (Vdss) was
observed. These results suggest that the 125I-rHSA dimer has
a better retention in the blood circulation, as compared with
125
I-native HSA.
Biodistribution of rHSA Dimer
The tissue distribution of 111In-labeled protein was
examined in the mouse (Fig. 4). The retention of the 111InrHSA dimer in the blood and plasma was confirmed whencompared with the 111In-native HSA. The accumulation of
the 111In-rHSA dimer in the kidney was significantly lower
only after 12 and 24 h. The accumulation of the 111In-rHSA
dimer in the liver was significantly higher at 12 h. The
accumulation of the 111In-rHSA dimer in the skin, muscle,
and fat was lower, with a significant decrease observed in theskin and muscle. In contrast, no significant difference wasobserved for the 111In-rHSA dimer in the urine and feces(Table II). The urine at 24 and 96 h from a mouseadministered with the 111In-rHSA dimer and 111In-native
HSA, respectively, was ultrafiltrated. More than 98% of thefiltrate was composed of low molecular substances under
30,000 in molecular weight, and the excretion of highmolecular weight protein was negligible. Ultrafiltration of
the plasma containing the 111In-rHSA dimer and 111In-native
HSA obtained at 1 h showed that more than 99% existed ashigh molecular weight protein.

Vascular Permeability of rHSA Dimer
The vascular permeability of the 125I-rHSA dimer was
evaluated using carrageenin-induced mouse paw edema as amodel system (Fig. 5). Carrageenin-induced mouse paw
edema is known to exhibit a biphasic inflammatory response(21). In this study, the biphasic response was confirmed bymeasurements of the increase in the thickness of the foot.
The vascular permeability of the 125I-rHSA dimer was low
between 0.5 and 120 h, especially at 0.5, 1, 4, 8, 24, 48, and
96 h where a significantly low permeability was observed
compared with that of 125I-native HSA. These results suggestthat the escape of HSA to the extravascular space during
inflammation can be reduced by dimerization.
Pharmacokinetic Analysis of rHSA Dimer
in Carrageenin-Air-Pouch Rats
Pharmacokinetics of 125I-labeled native HSA and therHSA dimer in carrageenin-air-pouch rats was evaluated bymeasuring the residual TCA-precipitable plasma radioactivity. Table III shows the results of an analysis of thepharmacokinetic parameters using a two-compartment
model. This model also showed an improvement in theretention of 125I-rHSA dimer in the blood. A significant
difference in half-life of the " phase could be observed; theAUC for the 125I-rHSA dimer was 1 1/2 times larger, whereas
the CLtot value was 2/3 times smaller, as compared with 125Inative HSA. The Vdss for the 125I-rHSA dimer showed asignificantly lower value than that of 125I-native HSA as well.
Urine and feces were collected at 24-h intervals from rats

Table II. Urinary and Fecal Excretion of Radioactivity After an Intravenous Administration of 111In-native HSA or 111In-rHSA Dimer
Urine (%ID)

111

In-native HAS
In-rHSA dimer

111

All values are mean T SD (n = 3).

Feces (%ID)

12 h

24 h

12 h

24 h

1.65 T 0.02
1.74 T 0.37

3.37 T 1.08
2.67 T 0.36

0.82 T 0.15
0.68 T 0.20

1.43 T 0.06
1.32 T 0.18

Matsushita et al.

888

Fig. 5. Vascular permeability of 125I-native HSA (filled bar) and 125IrHSA dimer (open bar) in mouse models of paw edema. A line graphshows the increase in footpad thickness. The data are the averagevalues of five to seven experiments (TSD). *p < 0.05, .p < 0.01 vs.
125
I-native HSA.

different from those used for blood collection kept inmetabolic cages (Table IV). According to the comparison
between rats administered with the same proteins, the airpouch model rat showed a greater decrease in the earlyexcretion in both 125I-native HSA and the 125I-rHSA dimer
than normal rats. In addition, the 96-h alveolar activity of rats
kept in the metabolic cage was determined. No significant
difference in the weight of alveolar fluid was found between
125
I-native HSA and the 125I-rHSA dimer (Table V).

DISCUSSION
Intravascular fluid therapy is a common critical careintervention. Many fluids have been studied for use inresuscitation, and these include isotonic sodium chloride
solution, lactated Ringer solution, hypertonic saline, albumin,
purified protein fractions, freshly frozen plasma, hetastarch,
pentastarch, and dextran 70. In spite of the many theoreticalbenefits of human albumin administration in critically illpatients, there has been little evidence to support its
widespread clinical use. Previous systematic reviews haveproduced conflicting results regarding the safety and efficacyof the use of albumin (2,22). The recently reported saline vs.
albumin evaluation study has provided conclusive evidence
that 4% albumin is as safe as saline for resuscitation,although no overall benefit of albumin use was reported(3,23). Albumin solutions for resuscitation may still bewarranted in certain highly selected patient populations suchas liver transplant patients and burn patients.

Albumin plays an important role in the circulation by
generating an inward oncotic force that counteracts theoutward capillary hydrostatic force. Without albumin, plasma
volume cannot be maintained and massive interstitial edema
results. In burn shock, in which the effective circulation is
reduced, albumin and extracellular fluid are rapidly shiftedfrom the plasma and sequestered in interstitial fluids. It is
commonly thought that the permeability of the capillarymembranes is pathologically increased in states of shock.
Burn injury is also characterized by a dramatic increase incapillary protein permeability. When shock does not develop
in small burns, or is prevented by fluid therapy, the rate ofprotein extravasation may increase to up to 100 times thenormal rate. Mechanisms that regulate plasma albumin
content are the transcapillary escape rate of albumin(TERalb), lymphatic albumin return, and the albumin synthesis-to-degradation ratio. TERalb measures the whole bodyrate at which albumin leaves the vascular space. It is
controlled by macrovascular and microvascular factors including plasma albumin concentration, plasma atrial natriuretic peptide concentration, capillary filtration coefficient,capillary hydrostatic pressure, interstitial fluid hydrostaticpressure, interstitial fluid colloid osmotic pressure, andinterstitial fluid albumin concentration (24,25).
Albumin leaves the circulation via several mechanisms,
which vary with the tissue involved. In organs havingsinusoids, i.e., liver and bone marrow, plasma can passthrough large gaps in the endothelium. Some other organs
have fenestrated endothelia that allow unimpeded passage;
the pancreas, small intestine, and adrenal gland are
examples of these. Collectively, these convection mechanisms account for about 50% of albumin transport from thecirculation. The transport may be described as taking placethrough two sets of parallel and cylindrical pores, one set
with diameters of 45Y60 Å and another with a diameter of200 Å. It has been shown that albumin, with an estimatedradius of 35.5 Å, and water do not share a common
pathway in crossing the endothelial monolayer, suggesting
the existence of a large pore pathway for albumin (26).
Results reported by Sejrsen et al. (27) indicate that the
predominant transcapillary transport mechanism for 131Ialbumin is compatible with transcapillary diffusion throughpores with an effective equivalent pore radius of 145 Å.
Hence, the extent of extravasation of albumin may be
reduced by increasing its molecular size.
In this study, we attempted to express an rHSA dimer byfusing two HSA molecules, containing two sets of the protein
sequence of 585 amino acids, linked with an amino acidlinker (GGGGS)2. As can be inferred from the intrinsic
fluorescence spectra and CD spectra, the structural characteristics of the rHSA dimer are almost identical to that ofTable III. Pharmacokinetic Parameters After an Intravenous Administration of 125I-native HSA or 125I-rHSA Dimer to Carrageenin-AirPouch Rats

125

I-native HAS
I-rHSA dimer

125

AUC (min x %ID/mL)

CLtot (2L/h)

Vdss (mL)

T1/2, " (h)103.1 T 5.3
164.0 T 10.1-

974.6 T 53.2
612.9 T 36.4-

22.38 T 1.01
19.47 T 1.09.

24.85 T 0.55
27.30 T 1.40*

All values are mean T SD (n = 5).
*p < 0.05, . p < 0.01, - p < 0.001 vs. 125 I-native HSA.

Biological Property of Recombinant HSA Dimer

889

Table IV. Radioactivity in Pouch Fluid After an Intravenous
Administration of 125 I-native HSA or 125 I-rHSA Dimer toCarrageenin-Air-Pouch Rats at 96 h
Pouch fluid (g)Radioactivity (%ID)

40.57 T 2.19
44.10 T 2.30

11.12 T 1.60
13.58 T 0.19

125I-native HSA
I-rHSA dimer

125

All values are mean T SD (n = 4).

native HSA. The pharmacokinetic analysis of the rHSA
dimer indicated that, in general, the rHSA dimer showed
longer intravascular residence time, as compared with native
HSA. (Fig. 3 and Table I). A biodistribution analysis of therHSA dimer generally showed a similar tendency with that of
the native HSA except for the fact that the extent ofaccumulation of the rHSA dimer in the spleen was muchgreater, whereas in the skin and muscle, the accumulation
was less than that of native HSA (Fig. 4). This observation isin agreement with the findings reported by Rennen et al.
(28), in which a rapid blood clearance with predominant
uptake by the liver and spleen was found for the largest
proteins (MW 669Y800 kDa), a relatively slow blood
clearance with relatively moderate uptake by the liver andspleen was seen for proteins of intermediate size (MW66Y206 kDa), and a rapid blood clearance with predominant
kidney uptake was found for smaller proteins (MW 2.5Y29
kDa). However, it is noteworthy that the blood retention
properties of the rHSA dimer were higher in comparison
with native HSA (Table I). In contrast, no significant
difference could be observed for the rHSA dimer and native
HSA in terms of excretion in the urine and feces (Table II).
HSA is not degraded by a specific organ but by manyorgans such as the liver, kidney, skin, and muscle. The bindingof albumin to the endothelial surface apparently initiates its
transcytosis via plasmalemmal vesicles and also increases
capillary permselectivity. Scavenger receptors interact with avariety of modified proteins, mediate their endocytosis anddegradation, and may play an important role in protein
catabolism and pathogenic processes such as atherosclerosis,
aging, and diabetes. Specific albumin binding to the surface ofthe endothelium initiates its transcytosis across the continuous
endothelium via noncoated plasmalemmal vesicles. Previous
studies have identified several putative albumin-binding
proteins (SPARC, gp60, gp30, and gp18). Two membrane-

associated proteins, gp30 and gp18, interact more strongly
with albumins that have been conformationally modified bychemical means or by surface adsorption to colloidal gold
particles than with native albumin. The gp30 and gp18 proteins
behave similarly to other known scavenger receptors, suggesting that gp30 and gp18 may mediate the high-affinity binding,endocytosis, and degradation of conformationally modified
albumins but not native albumin, whereas albondin mediates
native albumin binding, which significantly enhances its
transcytosis and capillary permeability (29,30).
On the other hand, the effect of an increase in vascularpermeability during inflammation on the vascular permeabilityof the rHSA dimer was evaluated using carrageenin-induced
paw edema in mice (Fig. 5). This carrageenin-induced
inflammation can be used to evaluate acute exudative stage
and chronic proliferative stage of carrageenin-air-pouch
inflammation in rats (31). Up to 120 h, the vascular permeability of the rHSA dimer was less than that of native HSA in
the model mice. In particular, significant difference was observed during the acute inflammation stage. In addition, thepharmacokinetics of the rHSA dimer was evaluated in carrageenin-air-pouch rats (Table III). Similar profile has beenobtained for the pharmacokinetics of the rHSA dimer in the
inflammation mouse model as in the normal rats. In comparison with native HSA, longer blood retention and lower
vascular permeability of the rHSA dimer were shown by thepharmacokinetic and vascular permeability analyses.
In contrast to our observations, McCurdy et al. (32)showed that a recombinant rabbit albumin (RSA) dimer
consisting of N-terminal hexahistidinyl tagRSA(C34A) joined
to G6 joined to RSA containing the C34A mutation in thatorder, from the N-terminus to the C-terminus, is more rapidly
cleared in vivo than wild-type and mutant C34A rabbit albumins. The authors concluded from their study that albumin
dimerization does not look promising as a tool to slow clearance. They deduced that the mechanism of the accelerated
clearance of recombinant rabbit serum albumin appears to
involve the reticuloendothelial system. On the other hand,
Komatsu et al. (33) has demonstrated the utility of an rHSA
dimer, which was cross-linked by the thiol group of Cys-34
with 1,6-bis(maleimido)hexane, and its synthetic heme hybrid
as an oxygen carrier displayed a longer half-life in the bloodstream and tissue distributions that were similar to rHSA. The
discrepancy between the studies may probably due to species
difference, as shown in the study by Sheffield et al. (34).

Table V. Urinary and Fecal Excretion of Radioactivity (%ID) After an Intravenous Administration of 125I-native HSA or 125I-rHSA Dimer
to Normal or Carrageenin-Air-Pouch Rats
Excretion of radioactivity (%ID)
125

125

I-native HSA

NormalTime (h)
0Y24
24Y48
48Y72
72Y96

I-rHSA dimer

Air pouchNormalAir pouch

Urine

Feces

Urine

Feces

Urine

Feces

Urine

Feces

39.7 T 0.8
25.9 T 3.2
13.0 T 0.7
6.1 T 0.3

1.54 T 0.42
1.96 T 0.44
1.03 T 0.21
0.52 T 0.05

43.5 T 4.7
20.7 T 2.2
8.5 T 0.5
5.1 T 0.3

1.58 T 0.54
1.69 T 0.36
0.54 T 0.15
0.40 T 0.01

35.0 T 1.5
19.7 T 0.5
11.2 T 0.6
7.2 T 0.4

1.67 T 0.35
1.34 T 0.20
0.79 T 0.24
0.47 T 0.01

44.8 T 2.0
18.2 T 1.6
8.04 T 0.6
3.6 T 0.4

1.51 T 0.41
1.22 T 0.12
0.53 T 0.13
0.26 T 0.02

All values are mean T SD (n = 4).

Matsushita et al.

890
Indeed, the clearance of albumin depends on various
factors including its degradation by a variety of organs, transcapillary escape, filtration and reabsorption, and lymphaticdrainage, just to name a few. These pathways in turn are also
influenced by the physicochemical state of the protein, such asits charge and conformation. In skin, the clearance for themost neutral modified albumins and cationic albumins wasfound to be 20 and 80% greater than that for native albumin,
respectively. In skeletal muscle, the clearance for the mostneutral modified albumins and cationic albumins was found to
be 50 and 150% greater than that for native albumin, respectively. This clearly shows that charge affects the transvasculartransport of albumin (35,36). In this study, the fusion has been
carried out through the N- and C-terminals. Since no conjugation of any of the amino acid residues has taken place, thecharge of the rHSA dimer should be the same as the native
HSA (monomer albumin). In addition, environmental factors
such as the presence of nitric oxide have also been reported toinfluence the extravasation of albumin (37).
Collectively, an increase in the molecular size of HSA
probably prevents or retards its convection movement throughpores with diameters of 45Y60 Å, as observed by an increase in
AUC and a decrease in the volume of distribution as well as
the clearance of the dimer. Meanwhile, since the dimer
exhibits structural features similar to those of native albumin,
we hypothesize that transvascular movement of the rHSA
dimer takes place via the same route, i.e., gp60, as evidenced
by the similarity in tissue distribution profile. However, further research must be carried out to validate such speculation.
On the other hand, since HSA is an endogenous protein,
it has been developed for use as a drug carrier due to its
excellent biological compatibility. A promising outcome has
also been reported using recombinant albumin as a synthetic
heme carrier protein in producing artificial blood (38,39). It isnoteworthy that albumin has recently been used as a carrier toprolong the blood retention properties of therapeutic proteinssuch as interferon and growth hormone. Through genetic
fusion technology, the therapeutic protein is fused to albuminand, as a result, the plasma half-life of the protein becomes
dependent on the half-life of albumin (40Y45). Fusion toalbumin offers several advantages. Albumin is the most abundant protein in mammalian plasma and one of the longest
lived. It lacks posttranslational modifications, with the exception of extensive disulfide bonding. If albumin can be fused inframe with a therapeutic protein as a single-chain polypeptide,
the novel protein may acquire the slow clearance profile ofalbumin, while retaining the activity important for clinical use.
On the other hand, although conjugation of polyethylene
glycol with the protein may also produce similar tendency,such approach involves modification of the internal aminoacid side chain, which may change the charge distribution and,hence, affect the stability of the protein. Albuferon (albumininterferon alpha) exhibits more antiviral activity at clinicallyachieved serum levels than standard interferon alpha or themodified interferons, Pegasys (peginterferon alpha-2a) and
Peg-Intron (peginterferon alpha-2b) (43).
Clinical and preclinical results to date demonstrate that
Albuferon is well tolerated, with adverse events that aretransient and mostly mild to moderate in severity, and exhibits
a robust antiviral activity, with a pharmacokinetic profile that
supports dosing at intervals of 2 to 4 weeks. This prolongationin half-life prevents the frequent administration of thetherapeutic protein. Hence, although albumin genetic fusion
technology is very promising, if the blood retention propertiesof albumin can be improved further, it is expected that suchalbumin will be of great clinical use as a new drug delivery
system material in addition to being a plasma expander.

ACKNOWLEDGMENTS
We wish to thank the members of the Gene Technology
Center in Kumamoto University for their important contributions to these experiments.

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